Fatty acids can be present in samples both in unesterified (free) and esterified forms (e.g., as glycerolipids, glycerophospholipids, sterol lipids and sphingolipids). The fatty acid content in a sample can be underestimated using analytical test methods that only test for free fatty acids. In some situations, the total fatty acid content can be more representative of the overall status of a biological system or the nutritional value of a food.
Current test methods for determining fatty acids require laborious and time-consuming procedures which negatively affect the sensitivity of detection. For example, gas chromatography-mass spectrometry (GC-MS) has been traditionally the technique of choice. Analysis by GC-MS, however, requires a multi-step procedure for the hydrolysis and derivatization of the fatty acids to fatty acid methyl esters, and a chromatographic separation. Alternatively, liquid chromatography-tandem mass spectrometry (LC-MS) has been used and allows for the direct measurement of both free and esterified fatty acids without the need for hydrolysis or derivatization. Yet, LC-MS still requires the labor intensive and time consuming chromatographic separation step. Supercritical fluid chromatography-mass spectrometry and other similar techniques has also been used, but these techniques suffer from the same requirement. Furthermore, any detailed spatial distribution of these species on a sample surface is unavailable using traditional sample preparation and extraction protocols.
The present disclosure relates to methods and apparatus for determining both free and total fatty acids which are less time consuming and resource intensive.